MicroRNAs (miRNAs) represent a class of endogenous small RNAs that sequence-specifically regulate target gene expression at the post-transcriptional level. In animals, miRNAs predominantly specify translational repression via binding to sites of imperfect complementarity in the 3' untranslated region of the target mRNAs. miRNA regulation has been linked to several human diseases including cancer and fragile X syndrome. In aim 1, I propose a biochemical strategy for systematic identification of the bona fide mRNA target segments recognized by miRNAs. The protein components of the miRNA effector complexes can be identified concurrently. The strategy is applicable to various human cells and tissues. In aim 2, I propose to develop cell-based assay systems recapitulating miRNA-mediated translational control in order to validate the targets and protein factors identified biochemically. I anticipate that comprehensive identification of miRNA targets will expand our knowledge of miRNA function and unravel more linkages between miRNA regulation and human diseases. In addition, identification of the components of the miRNA effector complexes will facilitate our efforts to elucidate the mechanism underlying miRNA regulation.